A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells

Duke University, Durham, North Carolina, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2005; 280(2):1156-64. DOI: 10.1074/jbc.M412006200
Source: PubMed


Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

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    • "In the ''lipid-dependent'' pathway myo-inositol is converted to phosphatidylinositol (PtdIns) by a phosphatidylinositol synthase (PtdIS), PtdIns is then sequentially phosphorylated to produce PtdIns(4,5)P 2 . This molecule is the substrate of a PtdIns-specific phospholipase C activity that releases Ins(1,4,5)P 3 , a molecule central to signal transduction (York et al. 1999; Odom et al. 2000; Fujii and York 2005; Seeds et al. 2004). In the ''lipid-independent'' pathway myoinositol phosphate is sequentially phosphorylated to lead to the formation of phytic acid (reviewed by Raboy and Bowen 2006). "
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    ABSTRACT: Phytate (myo-inositol hexakisphosphate), the major form of phosphorous storage in plant seeds, is an inositol phosphate compound poorly digested by humans and monogastric animals. A major goal for grain crop improvement is the reduction of its content in the seed to improve micronutrient bioavailability and phosphorus utilisation by humans and non-ruminant animals, respectively. We are interested in lowering phytic acid in common bean seed and to this goal we have undertaken a two-strategy approach: the isolation of mutants from an EMS mutagenised population (Campion et al. 2009) and the identification of genes coding for candidate enzymes involved in inositol phosphate metabolism for future targeted mutant isolation and/or study. In this paper we report data referred to the second approach and concerning the isolation and genomic organisation of Phaseolus vulgaris genes coding for myo-inositol 1-phosphate synthase (PvMIPSs and PvMIPSv), inositol monophosphatase (PvIMP), myo-inositol kinase (PvMIK), inositol 1,4,5-tris-phosphate kinase (PvIPK2), inositol 1,3,4-triphosphate 5/6-kinase (PvITPKα and PvITPKβ) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1). All these genes have been mapped on the common bean reference genetic map of McClean (NDSU) 2007 using a virtual mapping strategy. Bean markers, presumably associated to each gene of the phytic acid pathway, have also been identified. In addition, we provide a picture of the expression, during seed development, of the genes involved in phytic acid synthesis, including those such as MIK, IMP and IPK2, for which this information was lacking. KeywordsGene expression- Myo-inositol hexakisphosphate-Nutritional quality-Phosphorous-Phytate-Seed
    Molecular Breeding 04/2010; 25(3):453-470. DOI:10.1007/s11032-009-9344-1 · 2.25 Impact Factor
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    • "METABOLISM OF IP AND PP-IP SPECIES IN PLANTS, FLIES AND MAMMALS The genes involved in IPs and PP-IPs synthesis are evolutionary conserved from yeast to man, however in mammals there are two additional classes of kinase as well as gene duplications for several of the six kinases (Figure 1). In eukaryotic species examined to-date, all appear to require a Ipk2 homolog (also known as inositol phosphate multi-kinase, IPMK) and a Ipk1 homolog to produce IP 5 and IP 6 (Chang et al., 2002; Frederick et al., 2005; Fujii and York, 2005; Nalaskowski et al., 2002; Saiardi et al., 2001; Seeds et al., 2004; Stevenson-Paulik et al., 2005; Stevenson-Paulik et al., 2002; Verbsky et al., 2005a; Verbsky et al., 2005b; Verbsky et al., 2002; Xia et al., 2003). However, additional IPKs are identified in higher organisms, which represent alternative biosynthetic routes to IP 6 . "

    Advances in enzyme regulation 12/2009; 50(1):324-37. DOI:10.1016/j.advenzreg.2009.12.002
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    • "Heterologous expression of Arabidopsis thaliana, Drosophila melanogaster and Rattus norvegicus IPK2/IPMK gene products in ipk2 deficient yeast restore IP 4 /IP 5 synthesis activity (Fujii and York, 2005; Seeds et al., 2005, 2004; Stevenson-Paulik et al., 2005, 2002; Xia et al., 2003). Studies in which yeast and rat GFP-Ipk2 were individually overexpressed in Rat-1 cells demonstrated that both resulted in a 4-fold elevation in cellular IP 5 levels despite the fact they differentially localized to cytoplasmic and nuclear compartments, respectively (Fujii and York, 2005). These data indicate that localization does not appear to be required for metabolic function. "
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    ABSTRACT: Our laboratory studies the biology and enzyme regulation of inositol signal transduction pathways, which are activated in response to a wide range of stimuli. As a six-carbon cyclitol, inositol and its numerous phosphorylated derivatives efficiently generate combinatorial ensembles of signaling molecules. Through the cloning and characterization of inositol polyphosphate kinases (IPK), novel roles for inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6) and inositol pyrophosphates (PP-IPs), have been identified. Studies have linked the IPKs and their inositide products to the regulation of nuclear processes including gene expression, chromatin remodeling, mRNA export, DNA repair and telomere maintenance. Analysis of IPK knockout animals has revealed a role for production of IPs in regulation of embryogenesis and organism development. The discoveries of the IPK proteins and their connection to nuclear signaling have generated significant interest in the field. Furthermore, they have provided interesting clues into the evolution of inositide-signaling pathways. Ipk2/IPMK and IPS/IP6K family members are conserved from yeast to man. In contrast, the IP3 3-kinase (ITPK) branch is observed in selected metazoans and not in plant or fungi. This may imply that Ipk2 and IPS activities evolved first among the group. The promiscuity of the Ipk2 protein further supports this notion and may provide the cell with a means to generate many IP species in a genetically economical fashion. Studies of yeast inositide signaling reveal that these simple eukaryotes do not have an IP3 receptor in their genome and do not utilize diacylglycerol to activate protein kinase C. Thus, it appears that the canonical "text book" aspects of inositide-signaling pathways are not conserved throughout eukaryotic evolution. In light of the conservation of Ipk2/IPMK, Ipk1 and IPS/IP6K pathways from yeast to man it is interesting to speculate that a primordial role of phospholipase C-induced, IPK-dependent inositide signaling was to regulate nuclear processes. As calcium and PKC signaling evolved in metazoans, these may have greatly enhanced signaling capabilities. Recent studies demonstrating an essential role for IP5, IP6 and possibly PP-IP production in metazoan development highlight the importance of IPK signaling in cellular responses in metazoans. With these thoughts in mind, we eagerly await future studies aimed at further elucidating how these signaling codes participate in developmental processes and the control of gene expression, mRNA export, and DNA metabolism.
    Advances in Enzyme Regulation 02/2007; 47(1):10-25. DOI:10.1016/j.advenzreg.2006.12.019
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